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SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
42
RESEARCH ARTICLE
Multiple Cytokine Inhibition through Specific Procyanidin – Enriched
Plant Extracts: Implications for the Treatment of Psoriasis, Eczema and
Dermatitis
Ravi Shrivastava1*, Nathalie Cucuat
2, Christiane Shrivastava
3, Monika Rousse
4
1,2,3,4VITROBIO Research Institute, ZAC de Lavaur, 63500 Issoire, France.
Received 16 May 2013; Accepted 28 May 2013
* Corresponding author: [email protected] Tel: +33 4 73 55 05 05; Fax: +33 4 73 55 00 11
ABSTRACT
Psoriasis, Eczema and Dermatitis (PED) disorders involve abnormally rapid, excessive
epidermal cell growth, often followed by secondary infections. Over 20 cytokines, small
protein molecules, are thought to act as growth factors. As none of the currently available
topical treatments targets all the cytokines implicated in PED, their efficacy is extremely
limited, while the systemic administration of immuno-modulating or anti-mitotic drugs has a
very poor benefit/risk ratio. Our objective was to identify all the cytokines responsible for
PED and to neutralize them using semi-specific procyanidin fractions of plant tannins in
vitro. 29 purified cytokines considered to be very probably implicated in PED, were applied,
either singly or in association, on human epidermal cell cultures to measure their effects. The
cytokine association (VB-cytokines) inducing maximum growth was pre-incubated with
individual plant procyanidins (PCDs), and the most active PCD association, inducing over
90% VB-cytokine neutralization, was identified. Among 29 active cytokines, we identified 11
cytokines operating in synergy to stimulate excessive, uncontrolled epidermal cell growth. 12
among 134 PCDs, having strong affinities for those proteins, proved capable of blocking all
VB-cytokines and normalizing keratinocyte growth. Using specific plant PCDs to inactivate
all PED-involved cytokines is a novel and safe scientific approach to treat PED. Keywords: Cytokine. Dermatitis. Eczema. Psoriasis. Tannins.
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
43
INTRODUCTION
Affecting 2-15% of the population,
Psoriasis, Eczema, and Dermatitis (PED)
are common immune-based inflammatory
skin disorders, related to excessive and
abnormal cell growth in certain areas of
the skin (Fridman et al. 2011). Psoriatic
plaques, red and scaly patches on the skin
surface generally caused by Psoriasis, are
inflammation areas caused by excessive
cell growth. Frequently occurring on the
elbows and knees, but also on the scalp,
hand palms, feet soles, and genitals, those
plaques may become infected due to the
weakening of the skin tissue (Roberson
and Bowcock 2010). In contrast to
Psoriasis, Eczema is more likely to be
found on the flexor aspect of joints.
Eczema lesions are skin rashes
characterized by symptoms which include
redness, swelling, itching and dryness,
sloughing, flaking, blistering, cracking,
oozing, or bleeding (Biagini-Myers and
Khurana-Hershey 2010). Dermatitis is also
linked to anomalous, excessive epidermal
cell production, with weakening of the
affected areas and possible secondary
infections (Brook 2002). The skin
becomes red, flaky, very itchy, thick and
dry, and may develop bacteria-infected
papules (Boguniewicz and Leung 2010).
Seemingly distinct, those conditions share
the same basic cellular physiopathology,
involving a common factor. While their
exact causes have yet to be clearly defined,
based on patients’ history and genome-
wide linkage scans, which have identified
multiple loci on chromosomes 1q21, 3q21,
17q25 and 20p12 in such patients
(Zeeuwen et al. 2008) (Capon et al. 2012),
PED disorders are largely attributed to
genetic factors. Auto-immunity, immune-
modulation, allergic reaction, stress, and
other causes may also be involved, but
excessive skin cell growth, in correlation
with the presence, at the lesion site, of
cytokine-secreting cells (neutrophil
granulocytes, macrophages, lymphocytes)
(Bernard et al. 2012) (Boguniewicz and
Leung 2011) and cytokines (Nograles et al.
2008), can be observed in all PED
conditions. Cytokines are small, soluble
proteins, synthesized by the immune
system, and their role includes
communication and regulation of cellular
activities and cell growth functions. Some
cytokines, e.g.: EGF, KGF (Keratinocyte
Growth Factor), tumor necrosis factor-
alpha (TNF alpha), IL-6 (interleukin-6),
IL-22, IL-1B, IL-2, IL-3, IL-4, IL-5, IL-
10, IL-12, granulocyte-macrophage
colony-stimulating factor (GM-CSF), stem
cell factor (SCF), granulocyte-macrophage
colony stimulating factor (rhGM-CSF),
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
44
thrombopoietin, IL-23, platelet derived
growth factor (PDGF), and fibroblast
growth factor (FGF), are thought to act as
key growth factors to stimulate cell
growth, while some other cytokines, such
as macrophage migration inhibitory factor
and T-helper 1 (Th1) cytokine interferon-
gamma (IFN-γ), are supposed to block cell
growth (Ilkovitch 2011) (Nestle et al.
2009). The growth factor-like cytokines
are apparently present in very high
concentrations in the PED lesions,
inordinately promoting cell growth. As a
result, the epidermis becomes loosely
attached, and prone to microbial
contamination, leading to the appearance
of symptoms typical of PED. The best
treatment solution resides in stopping
excessive cell growth by normalizing
topical cytokine activity. Suppressing the
cytokine-secreting, immune-modulating
cells at the lesion site is one way to obtain
cell growth regulation. Unfortunately, a
medicine capable of blocking the functions
of all those immune cells selectively and
topically has not been conceived yet. And
even if a mechanism were found to block
these cells locally, newly synthesized
immune cells would keep on circulating
and infiltrating into the lesion, limiting the
treatment’s efficacy. Alternatively,
cytokine functions could be blocked by
using oral or intravenous preparations.
Unfortunately, the cytokines involved are
of multiple types, whereas all currently
available treatments are very specific, each
neutralizing solely one or two molecules,
so that significant therapeutic efficacy
remains out of reach (Mease 2006), for
instance new drugs like adalimumab,
infliximab and etarnercept block only
TNF-α; kineret blocks IL-1; protopic
inhibits IL-2; efalizumab antibodies only
bind LFA-1 antigen; and fusion protein
alefacept only blocks CD2 receptors on T-
cells (Laws and Young 2012). Moreover,
the systemic activity of these drugs
induces multiple, severe side effects (Roé
et al. 2008). Another therapeutic approach
would be to employ a topical multiple
cytokine inhibitor capable of neutralizing
all the cell growth-stimulating cytokines at
once, so as to normalize the PED
accelerated keratinocyte growth rate
without altering systemic immune
functions. But the cytokines involved in
PED have not all been discovered yet, and
the scientific information available is too
highly variable to target anti-cytokine
research at a defined group of cytokines.
Therefore, the aim of this in vitro research
was to identify all the specific cytokines
responsible for the accelerated cell growth
in PED lesions and seek means to
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
45
neutralize only those particular cytokines,
as a safe treatment for PED. Considering
that all cytokines and growth factors are
proteins, we postulated that they could be
neutralized by specific tannins. Tannins
are abundant in the plant kingdom and are
known for their affinity to bind with a
wide range of protein molecules. The
procyanidin (PCD) fraction of tannins
contains big phenolic compounds with
multiple-structure units and may have
selective protein-binding properties (Le
Bourvellec and Renard 2012).
This
multiple structure of tannins confers them
the capability to form strong hydrogen
bonds with various cytokines. In vitro
methods were employed to identify and
ultimately neutralize these cytokines as
new therapeutic approach for PED.
MATERIALS AND METHODS
In Vitro Model of Human Skin Cultures
Normal human keratinocytes, in the form
of a multi-layered, reconstituted epidermis
presenting a histological structure identical
to in vivo human skin, were used to mimic
a PED skin model in vitro. Fresh
epidermis cultures were purchased from
EPISKIN-SNC (Lyon, France). Each
epidermal unit consisted of an organotypic
culture made of adult human
keratinocytes, becoming stratified
epidermis after 10-14 days of culture, with
12-15 cell layers at maturity. Cells were
prepared from scrapings of normal human
skin in modified MCDB-153 cell culture
medium (Sigma-Aldrich, France),
dissociated enzymatically and seeded onto
a collagen (type I) matrix-coated
polycarbonate filter capable of absorbing
culture medium. Each filter was placed on
a sterile sponge, kept in a 6-well tissue
culture plate. Only the sponge was
immersed in culture medium (3ml MCDB-
153 with 10% fetal calf serum) so as to
keep the epidermis in an air (outer) to
liquid (lower) position, like human skin in
vivo. Cultures were incubated at 37°C in a
5% CO2 atmosphere. Under normal
conditions (replacing culture medium
every 5-6 days), the epidermis is
composed of 4-5 cell layers after 1 week
of culture, 7-8 after 2 weeks, and 8-12
after 4 weeks. Addition of non-cytotoxic
concentrations of growth factors or
cytokines to the culture medium increases
the speed of keratinocyte growth, resulting
in uncontrolled cell proliferation.
Excessive cell growth reduces the amount
of tissue culture medium and nutrition
available, causing keratinocyte sloughing
and cell death, similarly to PED skin
lesions in vivo. Cell growth can be
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
46
quantified to evaluate the effect produced
by the test products.
Plant Extract PCD Preparation
Procyanidin-rich plant extracts were
prepared from 222 tannin-rich plants using
methods described by B. Giner-Chavez et
al. (1999). Briefly, the primary tannin-rich
plant extracts were obtained with an
aqueous organic solvent (70% acetone and
30% water). Those extracts were then
successively passed through Sephadex
LH-20 columns by progressively
increasing the volume of methanol (60 x
88.5 cm), and the intended fractions were
eluted to produce a dry solid, which was
identified by mass spectrometry. The
extracts mainly contained procyanidin
(epicatechin–catechin) B1, B2, B3 and C1
fractions, between 60% and 80%
depending on the plant part used. The
concentrated plant extract was then dried
by atomization to obtain a PCD-rich plant
powder which was stored at 4°C for later
use. In preliminary experiments, 134 PCD
extracts were selected based on their lack
of cytotoxicity, and their cytokine-binding
properties. After initial screening, only 32
extracts, showing some anti-cytokine
activity, were retained for further
evaluation. The key plant PCDs tested
were E. purpurea (aerial parts), Mimosa
tenuiflora (bark), Aesculus hippocastanum
(aerial parts), Salvia officinalis (flowering
aerial parts), Alchemilla vulgaris (aerial
parts), Centella asiatica (leaves), Camellia
sinensis (aerial parts), Acacia catechu
(bark gum), Vitis vinifera (leaves),
Prunella vulgaris (leaves), Tanacetum
parthenium (aerial parts), Calendula
officinalis (flowering aerial parts), Oak
bark (Quercus alba, Quercus rober), and
fruit extracts of Sambucus nigra,
Vaccinium myrtillus, and Vaccinium
macrocarpon. For all experiments, PCD-
enriched plant extracts were tested at a
concentration of 10 mg/ml when used
singly, or 5 mg/ml when used in any
association.
Cytokine Selection
All cytokines cited in the literature for
direct or indirect influence on epidermal
cell growth were screened. 29 purified,
recombinant test cytokines with known
biological activity and insignificant
endotoxin levels were purchased from
Peprotech (France) and tested: EGF
(Epithelial Growth Factor also called
Insulin-like Growth Factor-II,
Somatamedin A); PDGF-AA (Glioma-
derived growth factor - GDGF,
Osteosarcoma-derived Growth Factor -
ODGF); TGF-α (Transforming Growth
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
47
Factor-α, Sacroma growth factor, TGF-
type I, ETGF); TGF-β (Transforming
Growth Factor-beta1, Differentiation
inhibiting factor, Cartilage-inducing
factor); TNF-α (Tumor Necrosis Factor,
TNFSF2, Cachectin, Differentiation-
inducing factor-DIF, Necrosin, Cytotoxin);
TNF-β (Tumor Necrosis Factor-beta,
TNFSF1, Lymphotoxin-alpha); FGF-21
(Fibroblast Growth Factor-23);
KGF/FGF-7 (Keratinocyte Growth
Factor, Fibroblast Growth Factor-7,
HBGF-7); M-CSF (Macrophage Colony
Stimulating Factor, CSF-1, MGI-IM);
GM-CSF (Granulocyte/Macrophage
Colony-Stimulating Factor, CSF-2, MGI-
1GM, pluripoietin-α); HB-EGF (Heparin
Binding EGF-like growth factor, HBEGF,
Diphteria toxin receptor, DTR); IL-1α
(Hematopoietin-1, Lymphocyte-activating
factor-LAF, Endogenous Pyrogen-EP,
Leukocyte Endogenous Mediator-LEM),
Mononuclear Cell Factor-MCF; IL-1β
(Catabolin, Lymphocyte-activating factor-
LAF, Endogenous Pyrogen-EP, Leukocyte
Endogenous Mediator-LEM), MCF
(Mononuclear Cell Factor); IL-1β
Interlulin - 1 Beta, Catabolin,
Lymphocyte-activating factor-LAF,
Endogenous Pyrogen-EP, Leukocyte
Endogenous Mediator-LEM, Mononuclear
Cell Factor-MCF); IL-2 (T-cell growth
factor-TCGF, Aldesleukin); IL-3 (MCGF-
Mast cell growth factor, Multi-CSF,
HCGF, P-cell stimulation factor); IL-4
(BCGF, BCDF, B cell stimulating factor-
BSF-1); IL-6 (26 kDa protein, IFN-β2, B
cell differentiation factor-BCDF, BSF-2,
HPGF, HSF, MGI-2); IL-10 (B-TCGF,
CSIF, TGIF); IL-11 (AGIF-Adipogenesis
inhibitory factor); IL-12 (NKSF, CTL
maturation factor-TCMF, Cytotoxic
lymphocyte maturation factor-CLMF,
TSF); IL-13 (NC300-Human, P600-
Murine); IL-15 (IL-T); IL-16 (LCF-
Lymphocyte Chemoattractant Factor); IL-
17D (IL-27); IL-17F; IL-19 (Melanoma
differentiation association like protein);
IL-22 (IL-TIF); FGF-23 (Fibroblast
Growth Factor-23); and SCF (Stem Cell
Factor, c-Kit Ligand, Mast Cell Growth
Factor-MGF, Steel Factor). The non-
cytotoxic yet biologically active
concentration used for each cytokine is
indicated in the result tables.
Test Product Exposure
Only young, exponentially growing
epidermis cultures, presenting 3-4 cell
layers (3-5 days old) were used for the
experiments. At the start of each
experiment, standard growth medium was
replaced by fresh, serum- and cytokine-
free medium, under sterile conditions. 50
µl of test product containing appropriate
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
48
concentrations of the test substance were
then added to the culture medium, and
cells were incubated at 37°C in 5% CO2
atmosphere for a period of 4 extra days to
quantify cell growth. Minimum 3 cultures
were used to test each concentration.
Untreated cells served as negative
controls. During preliminary experiments,
cells were first exposed to all plant
extracts, cytokines, and PCDs, to
determine the maximum non-cytotoxic
concentration of each test product to be
used for further experiments. Cell growth
was determined using cell viability MMT
test and histological analysis.
Cell Viability Measurements
To quantify live cells, the polycarbonate
filter containing cultured epidermis was
removed and cut vertically into equal
halves. One half was stained with MTT
stain to check cell viability as described by
Osborne and Perkins (1994). Live cells
stain red due to mitochondrial
dehydrogenase activity. Red color
intensity was evaluated by introducing 100
µl of MTT-stained cell culture solution
into 96-well tissue culture microplates and
by measuring the optical density (OD) at
650 nm (Dynatec MR 400). The mean OD
of untreated cultures served as control
reference to which the mean OD values of
treated cultures were then compared.
Results were expressed on a scale of 0
(100% cell death) to 3, considering that the
OD of control cultures was 1
(corresponding to normal number of layers
of live cells).
Histological Cell Growth Analyses
The second half of the epidermis was fixed
in formaldehyde and stained with standard
Hematoxylin & Eosin (H&E) stain for
microscopic examination. In short, at the
end of the experimental period, each
second epidermis half-section was
immediately washed with PBS and fixed
in buffered 4% paraformaldehyde
overnight. Samples were then embedded in
a paraffin cassette, and the cassettes were
transferred into 70% ethanol and stored at
4°C for later use. For histological
evaluation, 5micron-thick vertical sections
of epidermis were cut (Microtom, Leica),
deparaffinised, rehydrated and stained with
H&E stain as described by Li et al (2011).
Cell growth in treated cultures was
evaluated and compared to the number of
cell layers in the negative control cultures
(usually around 7-8) which was scored as
1. Scores went from 0 to 3, 0 indicating
total cell death, 1 denoting a number of
cell layers identical to controls, 2
corresponding to a number of cell layers
around 12-14, and 3 indicating excessive
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
49
cell growth, with about 15-20 cell layers
and cell sloughing. This arbitrary 0-3 scale
was chosen because exact quantification is
difficult due to cell sloughing and overlap,
and it constitutes a useful tool to quantify
PED-type of cellular distribution. Scores
obtained for cell viability and cell
histology at each concentration (n=3
epidermis each) were averaged and
mutually compared to evaluate the effects
of the test products on cell growth.
Determination of the Cell Growth-
stimulating Properties of Cytokines
To determine keratinocyte growth-
stimulating properties, exponentially
growing, 3-5 days old epidermis was first
exposed to each cytokine (n=3) for a 4-day
period. Cytokine concentrations ranged
between 0.001 and 5 ng/ml, depending on
pre-determined biological activity of each
cytokine (see result tables for
concentrations used). After 4-day
incubation, cell growth scores obtained
through MTT staining (on a 0-3 scale) and
histological examination (on a 0-3 scale)
for each epidermal section were added and
averaged to a mean cell growth score. The
score obtained for negative control (non–
cytokine-exposed) epidermis was 1
(normal cell growth), and the percentage
of change in cytokine-exposed cells
compared to negative controls was
calculated. Cytokines accelerating cell
growth by over 60% were classified as
highly active; between 40 and 60%, as
moderately active; between 20 and 39%,
as slightly active; and below 20%, as
inactive. Highly or moderately active
cytokines were then associated with each
other to identify the group of cytokines
involved in triggering the uncontrolled,
excessive growth typical of PED, this
combination being named VB-cytokines.
Identification of Cytokine-neutralizing
PCDs
The VB-cytokines association was then
incubated with each individual PCD for 1
hour at 37°C to allow cytokine-PCD
interaction. VB-cytokine activity on
cultured epidermal cells was then
evaluated as explained above. Unexposed
epidermis served as negative control while
epidermis exposed only to VB-cytokines
served as positive control. Results are
expressed as percentage of change induced
in cell growth, compared to the positive
control. Individual plant PCDs capable of
inhibiting VB-cytokines-induced excessive
cell growth by more than 10% were then
combined to identify the key PCD
association capable of maximal
normalisation of VB-cytokines-induced
cell growth.
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
www.soushrutam.com ISSN 2320-5830
50
Statistical Analyses
Statistical comparisons were made using
Student's t test. When more than two
groups were compared, ANOVA was also
employed.
RESULTS
The measured effects of individual
cytokines on epidermal cell growth are
shown in Table 1.
Only 18 out of 29 cytokines demonstrated
cell growth-enhancing properties. EGF,
TNF- α , KGF-7, GM-CSF, IL - 1 α, IL-6,
and SCF enhanced cell production by over
60% compared to non–cytokine-exposed
cells. Moderate cell growth stimulation
(between 40 and 60%) was observed with
FGF-21, IL-17D, IL-22, and IL-23 while
PDGF-AA, TGF- α, TNF - β, M-CSF, HB-
EGF, IL-3, and IL-10 were less active as
cell proliferation was only increased by
20-40%. All results were statistically
significant compared to cell growth in the
negative control (p<0.005). These
cytokines were further evaluated in
association with each other to identify the
specific cytokine combination most highly
responsible for PED cell proliferation. All
other cytokines were considered inactive
as their cell growth-amplifying effects at
biologically active concentrations were
below 20%, and statistically not significant
(p>0.05) compared to negative controls.
These results prove that not all but only a
few cytokines (11/29) cause accrued
epidermal cell multiplication; however the
increase in cell growth observed in vitro is
relatively modest compared to cell growth
in PED lesions in vivo.
Effect of the Association of Moderately
and Highly Active Cytokines on Cell
Growth
As shown in Table 2, results indicate that
although all the highly or moderately
active cytokines conserve their cell
growth-enhancing properties, the effects
are not additive. EGF + TNFα + FGF-21 +
KGF-7, + GM-CSF + IL-1 α + IL-6 + IL-
17D + IL-22 + FGF-23 + SCF, all
identified as either moderately or highly
active cytokines, act in a synergistic
manner to accelerate epidermal cell
multiplication. Different associations of
these cytokines eventually promoted cell
growth more than their individual activity,
but maximum cell growth was induced
only when these 11 cytokines were all
present together in the culture medium.
This shows that each cytokine acts in
synergy with the others to amplify
keratinocyte growth.
SOUSHRUTAM
An International Research Journal of Pharmacy and Plant science
Volume 1(4), May/June 2013
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Table 1: Cell growth-promoting properties of individual cytokines at biologically active concentrations
between 0.001 and 5 ng/ml in the culture medium.
Sl.
No.
Test
Cytokine
Conc.
Used
Ng/Ml
Average Scores of Mtt and
Histology for Each Section
Mean Cell
Growth
score
% Change
EXP 1 EXP 2 EXP 3
CONTROL 0.0 1.05 1.0 0.95 1.0 ±0.05 0.0
1 EGF 0.1 1.65 1.80 1.70 1.72 ±0.08 + 72.0%
2 PDGF-AA 0.5 1.30 1.35 1.30 1.32 ±0.03 + 32.0%
3 TGF-α 0.1 1.25 1.35 1.20 1.27 ±0.08 + 27.0% *
4 TGF - β 0.025 1.05 1.10 1.20 1.12 ±0.08 + 12.0% *
5 TNF - α 0.025 1.80 1.65 1.70 1.72 ±0.08 + 72.0%
6 TNF - β 0.025 1.30 1.40 1.40 1.37 ±0.06 + 37.0%
7 FGF – 21 2.0 1.65 1.50 1.60 1.58 ±0.08 + 58.0%
8 KGF – 7 5.0 1.75 1.85 1.80 1.80 ±0.05 + 80.0%
9 M – CSF 1.0 1.45 1.35 1.30 1.37 ±0.08 + 37.0% *
10 GM – CSF 0.05 1.75 1.75 1.75 1.75 ±0 + 75.0%
11 HB – EGF 0.05 1.30 1.30 1.35 1.32 ±0.03 + 32.0%
12 IL - 1 α 0.001 1.80 1.80 1.65 1.75 ±0.09 + 75.0%
13 IL – 1 β 0.001 1.0 1.0 1.0 1.0 ±0 0.0%
14 IL – 2 0.05 1.0 1.0 0.95 0.98 ±0.03 8.0% *
15 IL – 3 0.05 1.20 1.25 1.15 1.20 ±0.05 + 20.0% *
16 IL – 4 0.1 0.90 0.95 0.90 0.92 ±0.03 - 8.0% *
17 IL – 6 0.05 1.70 1.70 1.80 1.73 ±0.06 + 73.0%
18 IL – 10 1.0 1.30 1.25 1.25 1.27 ±0.03 + 27.0%
19 IL – 11 0.1 1.0 1.0 1.0 1.0 ±0 0.0%
20 IL – 12 0.05 1.0 1.0 1.0 1.0 ±0 0.0%
21 IL – 13 0.05 1.0 1.0 1.0 1.0 ±0 0.0%
22 IL – 15 0.05 1.0 1.0 1.0 1.0 ±0 0.0%
23 IL – 16
121AA
0.05 0.85 1.0 0.95 0.93 ±0.08 - 7.0% *
24 IL – 17 D 0.05 1.55 1.45 1.60 1.53 ±0.08 + 53.0%
25 IL – 17 F 0.05 1.0 1.0 1.0 1.0 ±0 0.0%
26 IL – 19 0.05 0.95 1.05 1.0 1.0 ±0.05 0.0%
27 IL - 22 0.05 1.65 1.50 1.55 1.57 ±0.08 + 57.0%
28 FGF - 23 1.0 1.55 1.50 1.55 1.53 ±0.03 + 53.0%
29 SCF 1.0 1.70 1.80 1.75 1.75 ±0.05 + 75.0%
30+ OTHERS 0.01-1.0 <1.10 <1.10 < 1.10 <1.10 < 10.0%
ǂ Mean cell growth score represents the average score (with Standard Deviation) of 3 experiments obtained
after MMT and histological analyses. % cell growth change indicates % increase or decrease in cell growth
compared to the negative, unexposed control cultures. All experiments were conducted in triplicate. All cell
growth changes are statistically significant (p<0.005) compared to controls, unless indicated by an asterisk *.
SOUSHRUTAM
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Table 2: Cell growth-promoting properties of associations of highly active and moderately active
cytokines, at biologically active concentrations between 0.001 and 5 ng/ml in the culture medium.
Sl.
No.
Association of Cytokines
Tested
Epidermal Growth
Score
Mean
Growth
Score
% Change
C- NEGATIVE CONTROL 1.05 1.00 0.95 1.00 ±0.05 0.0
1 EGF + TNFα + FGF-21 1.75 1.65 1.80 1.73 ±0.08 + 73.33
2 EGF + KGF-7, + IL-1 α 2.05 2.10 1.90 2.01 ±0.1 + 101.67
3 EGF + FGF-23 + SCF 2.05 1.85 1.85 1.92 ±0.11 + 91.67
4 FGF-21 + KGF-7 + GM-CSF 2.30 2.00 2.20 2.17 ±0.15 + 116.67
5 EGF + IL-6 + IL-17D + IL-22 1.80 1.75 2.0 1.85 ±0.13 + 85.00
6 EGF + FGF-21 + KGF-7 2.05 2.25 2.30 2.20 ±0.13 + 120.00
7 EGF + TNFα + FGF-21 + KGF-
7 + GM-CSF
2.30 2.40 2.30 2.33 ±0.06 + 133.33
8 KGF + IL-6 + IL-17D + IL-22 1.90 1.95 1.90 1.92 ±0.03 + 91.67
9 KGF + FGF-23 + SCF 2.20 2.10 2.35 2.22 ±0.13 + 121.67
10 KGF-7 + GM-CSF 2.20 2.10 2.10 2.13 ±0.06 + 113.33
11 IL-6 + IL-17D + IL-22 1.80 1.95 1.70 1.82 ±0.13 + 81.67
12 EGF + TNFα + FGF-21 + KGF-
7, + GM-CSF + IL-1 α + IL-6 +
IL-17D + IL-22 + FGF-23 +
SCF
2.80 2.65 2.60 2.68 ±0.1 + 168.33
13 OTHER ASSOCIATIONS <1.50 <1.50 <1.50 <1.50 < +50
ǂ Mean cell growth score represents the average score (with Standard Deviation) of 3 experiments obtained
after MMT and histological analyses. % cell growth change indicates % increase or decrease in cell growth
compared to unexposed control cultures. All experiments were conducted in triplicate and all cell growth
changes are statistically significant (p<0.005) compared to controls.
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Table 3: VB-cytokine inhibitory effect of PCDs: either singly or in different associations of active PCDs.
Sl. No. Test Cytokine
And Plant
Extract Code
Mean Growth
Score
% Change Vs
Negative Control
% Change Vs
Positive
Control
C - NEGATIVE
CONTROLS
1.0 0.0 % OK
C + POSITIVE
CONTROLS
2.68 + 168.3% +168.3
1 AC 2.03 + 103 % -38.8
2 SO 2.05 + 105 % -37.6
3 VV 1.61 + 61 % -63.76
4 QR 2.38 + 138% -18.0
5 GB 2.06 + 106 % -37.01
6 V SP 1.43 + 43 % -74.4
7 SA 1.57 + 57 % -66.13
8 MT 2.32 + 132 % -21.56
9 EP 2.35 + 135 % -19.79
10 TP 2.27 + 127 % -24.54
11 CS 1.77 + 77 % -54.25
12 SN 1.50 + 50% -70.29
13 OTHERS
ASSOCIATION
14 AC + VV 1.83 + 83 % -50.68
15 AC + MC 1.78 + 78 % -53.65
16 AC + EP 1.55 + 55 % -67.32
17 AC + CS 1.55 + 55 % -67.32
18 VV + GB 1.61 + 61 % -63.76
19 MC + EP 1.52 + 52 % -69.10
20 MC + CS 1.65 + 65 % -61.38
21 EP + CS 1.35 + 35 % -79.20
22 VV + V SP 1.18 + 18 % -89.30
23 VV + CS 1.25 + 25 % -85.15
24 VV + SN 1.30 + 30 % -82.17
25 VV + V SP + CS 1.14 + 14% -91.68
26 VV + MT + SN 1.38 + 38% -77.42
27 SN + V SP + CS 1.22 + 22% -86.93
ǂ Cell growth score of unexposed and untreated negative control cultures was considered as 1 (100%
cell growth). Positive controls were exposed only to VB-cytokines. Change in cell growth is
expressed as % increase (+) or decrease (-) in cell growth mean score compared to the positive VB-
cytokine-exposed cultures. All values are means of at least 3 experiments and all values are
statistically significant compared to the positive controls (p<0.005).
1 = Acacia catechu; 2 = Salvia officinalis; 3 = Vitis vinifera; 4 = Quercus robur (Oak bark); 5 = Ginko
biloba; 6 = Vaccinium sp. (fruits); 7 = Salix alba; 8 = Mimosa tenuiflora; 9 = Echinacea purpurea; 10
= Tanacetum parthenium; 11 = Camellia sinensis; 12 = Sambucus nigra. Others = as given in the list
of plants tested.
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Cytokine-inhibiting Properties of Plant
PCDs
VB-cytokines were incubated with a fixed
concentration (10 mg/ml singly, or 5
mg/ml each when used in association) of
PCDs from more than 130 tannin-rich
plants for 1 hour, and epidermal growth
was then quantified. As shown in Table 3,
in negative controls, without addition of
VB-cytokines or PCDs, cell growth was
normal (scored as 1) with the epidermis
showing 6-7 cell layers. In VB-cytokines
positive control group, (VB-cytokines
only, no PCDs), the number of epidermal
cell layers was 17-19, with inordinate cell
sloughing, representing excessive cell
growth with mean values 168.3% higher
than negative controls. Pre-incubation of
the same VB-cytokines with individual
plant PCDs showed no statistically
significant changes in cell growth for 120
of 132 PCDs, indicating absence of
binding between these PCDs and the
cytokines. Only 12/132 PCDs decreased
the excessive cell growth by 15% to 74%
compared to positive controls (p<0.005).
The highest VB-cytokines inhibition was
observed with PCDs obtained from
Vaccinium sp. (Vaccinium macrocarpon,
V. Myrtillus) and Sambucus nigra, since
they each inhibited VB-cytokines-induced
keratinocyte proliferation by about 70%.
PCDs from Vitis vinifera, Salix alba, and
Camellia sinensis were also effective,
decreasing VB-cytokines-induced cell
multiplication by 50-60%. Results
obtained with PCD associations indicate
that V. vinifera or S. nigra with Vaccinium
sp. or C. sinensis prove extremely
effective in neutralizing VB-cytokines, as
mean excessive epidermal growth was
reduced by 80-91% compared to positive
controls. These results clearly show that
PCDs are highly specific with respect to
their cytokine binding and that only a few
PCDs have the structural configuration to
bind with the cytokine proteins.
DISCUSSION
Normal keratinocyte maturation cycle of
28-30 days is accelerated to 4-5 days in
PED-affected skin, resulting in poor cell
attachment, cell sloughing, secondary
infections and persistence of PED lesions
(Halprin 1972). PED-type skin diseases
are almost uniformly considered to result
from changes in the body’s immunological
functions, some immunological reactions
probably causing tissue damage and a
localized inflammatory zone on the skin
(Guttman-Yassky et al. 2011). When the
skin detects a danger signal, a cascade of
highly sophisticated interactions between
keratinocytes and “sentinel” immune cells
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is triggered to maintain skin homeostasis.
The inflammation and subsequent cell
regeneration are relayed by more than 20
or 30 different types of small
communication protein molecules, called
cytokines, each playing a specific role in
the skin damage repair process. For a
number of known or unknown reasons
(genetic, environmental, etc.), faulty
signals occur during the resolution of this
phenomenon and generate a cytokine-
mediated vicious circle, promoting chronic
inflammation, huge infiltration of immune
cells in the dermis and epidermis,
production of chemokines and growth
factor-like cytokines, altered
differentiation of keratinocytes,
uncontrolled and excessive cell
production, and development of skin
lesions classified as psoriasis, eczema or
dermatitis, depending on the location and
appearance (Guilloteau et al. 2010),
although the basic physiopathology of
those three conditions remains more or
less identical. Different authors discovered
the predominance of multiple types of
proteins and cytokines acting as growth
factors, such as IL-1 (IL-1α and β), IL-2
(IL-4, IL-13, IL-21), IL-4 (IL-4, IL-13),
IL-6 (IL-6, OSM, IL-31), IL-10 (IL-19,
IL-20, IL-22, IL-24), IL-17 (IL-17A and
IL-17F), IFN (IFNα, IFNγ), or TNF
(TNFα, TNFβ) family (Caruso et al. 2009),
but other cytokines involved in the
pathology have not yet been identified.
Extreme innate immune response
prompting cytokine production, epidermal
basal layer cell over-proliferation,
epidermal layer disruption, in conjunction
with mechanical damage from intense
pruritus and desquamation, contribute to
the more severe sequelae, including
chronic bacterial colonization (S. aureus)
and penetration of exogenous substances,
such as allergens, irritants, microbes,
pollutants, and even topical drugs, into the
PED lesions. There is, as yet, no treatment
which can normalize the body’s immune
functions to derail the uninterrupted
inflammatory trigger, recruitment of
immuno-modulating cells in the PED
lesion, and excessive production of growth
factor proteins, so as to normalize
keratocyte growth without severe side
effects (Chaudhari et al. 2001). Moreover,
modifying the body’s immunological
mechanisms might stop PED pathogenesis
but would equally affect all other
immunological functions of the body,
which may prove disastrous for the
patient’s health. Considering the absence
of a truly effective treatment for PED and
the side effects associated with the
immune-modulating or biological
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therapies currently employed, products
acting only topically may represent the
therapy of choice for treating PED. As
PED skin is damaged and therefore highly
permeable, topical treatment efficacy
should be facilitated. If the lesion is
smooth or keratinized, scraping the surface
just enough to render it permeable should
allow PCDs to reach the site of cellular
proliferation. Since the basic cause of PED
is excessive cell production and certain
cytokines act as growth factors to maintain
cell proliferation, it was important to
identify all cytokines specifically
implicated in PED so as to envisage a
treatment. As it is practically impossible to
control the local functions of immune-
modulating cells with a single chemical
molecule without affecting vital cellular
functions in other parts of the body, we
employed plant tannins as natural multiple
protein inhibitors to neutralize the growth-
promoting cytokines. Currently, only
methotrexate is commonly employed to
reduce cell growth in PED (Lee et al.
2012). This is an orally-administered anti-
cancer drug which stops cell growth in the
S-phase of the cell cycle. Unfortunately,
none of the anticancer drugs can
differentiate between normal and
pathological cells, and they equally affect
the growth of healthy cells. Consequently,
their benefit/risk and cost/risk ratios are
extremely poor, not warranting their use to
treat a non life-threatening disease like
PED. Moreover, such drugs have no effect
on cytokines, the main cause of
symptomatic manifestations of
immunological disorders (Le Bourvellec
and Renard 2012). It was therefore
essential to target only those cytokines
involved in PED cell growth, without
affecting the functions of other cytokines
which may actually be useful to repair
PED lesions. Results of in vitro studies
have shown that as many as 29 cytokines
possess skin cell growth-promoting
properties but only 11 of them act
synergistically to induce uncontrolled cell
proliferation, the basic cause of PED
disorders. Tannins and PCDs are very big,
highly branched, safe molecules having a
strong affinity for proteins. We observed
that allowing tannin–cytokine interaction
effectively blocks the biological activity of
cytokines, but that only a few PCDs have
the capacity to bind with the VB-
cytokines. We postulated that applying
topically a synergistic association of
selected cytokine-inhibiting plant PCDs
might curb cytokine-induced cell
proliferation, and we pursued this research
hypothesis to conceive a new therapeutic
strategy, focused on neutralizing the cause
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of excessive cell growth by targeting only
growth-factor-like cytokines rather than
using non-specific systemic therapies or
interfering with immunological cellular
functions. Although further in vivo and
clinical trials are recommended to either
confirm or refute this hypothesis, the
specific blockage and inactivation of
cytokines responsible for epidermal cell
proliferation offers a new alternative for
the treatment of PED.
Acknowledgements & Declaration of Interests
This research was entirely carried out at, and supported by: VITROBIO, Research Institute,
ZAC de Lavaur, 63500, Issoire, France, without any other sponsor. The authors have full
control of all primary data, and were involved in the acquisition, analysis and interpretation
of data.
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Conflict of Interest statement
The Authors report no conflict of Interest.